The present invention relates to fluorescent dye compounds, and to conjugates and uses thereof. The invention also relates to fluorescent polynucleotide conjugates having improved electrophoretic mobilities.
The analysis of complex mixtures of polynucleotides is important in many biological applications. In many situations, it is necessary to separate components of such mixtures to detect target polynucleotides of interest, to determine relative amounts of different components, and to obtain nucleotide sequence information, for example.
Electrophoresis provides convenient methods for analyzing polynucleotides. Typically, polynucleotides can be separated on the basis of length, due to differences in electrophoretic mobility. For example, in a matrix such as crosslinked polyacrylamide, polynucleotides typically migrate at rates that are inversely proportional to polynucleotide length, due to size-dependent obstruction by the crosslinked matrix. In free solution, polynucleotides tend to migrate at substantially the same rates because of their substantially identical mass to charge ratios, so that it is difficult to distinguish different polynucleotides based on size alone. However, distinguishable electrophoretic mobilities can be obtained in free solution using polynucleotides that contain different charge/mass ratios, e.g., by attaching to the polynucleotides a polymer or other chemical entity having a charge/mass ratio that differs from that of the polynucleotides alone (e.g., see U.S. Pat. No. 5,470,705).
When different polynucleotides can be separated based on length or molecular weight, detection can usually be accomplished using a single detectable label, such as a radioisotope or fluorophore. However, in complex mixtures or when different-sequence polynucleotides have similar or identical mobilities, it is preferable to use two or more detectable labels to distinguish different polynucleotides unambiguously.
In DNA sequencing, it is now conventional to use two or more (usually four) different fluorescent labels to distinguish sequencing fragments that terminate with one of the four standard nucleotide bases (A, C, G and T, or analogs thereof). Such labels are usually introduced into the sequencing fragments using suitably labeled extension primers (dye-primer method) or by performing primer extension in the presence of nonextendable nucleotides that contain unique labels (Sanger dideoxy terminator method). Electrophoresis of the labeled products generates ladders of fragments that can be detected on the basis of elution time or band position.
Under sieving conditions in crosslinked or non-crosslinked matrices, shorter poly-nucleotide fragments migrate more rapidly than longer fragments. Usually, the inter-band spacing and migration rates of fragments decrease gradually in proportion to increasing length. However, anomalous migration patterns can occur due to sequence-dependent secondary structures within fragments, even in the presence of denaturing agents such as urea. For example, poly-G segments often cause band compression that make sequence determination of these regions difficult. Compressed band regions can often be resolved using nucleotide analogs such as dITP (2xe2x80x2-deoxyinosine-5xe2x80x2-triphosphate) or 7-deaza-dGTP in the extension reaction instead of dGTP, or by sequencing the complementary strand.
Anomalous migration patterns may also occur for polynucleotide fragments that contain a detectable label, due to interactions between the label and one or more bases in the polynucleotide. Such interactions can be particularly problematic when the interactions are sequence-dependent, so that different-sequence fragments having the same-lengths may have significantly different mobilities. This phenomenon can be inconvenient for sequencing, especially in automated sequencing methods. Accordingly, there is a need for labeled compounds and methods of use to improve electrophoretic performance.
In one aspect, the present invention provides a conjugate comprising a dye-labeled nucleobase of the form: (1) B-L-D, wherein B is a nucleobase, L is an anionic linker, and D comprises at least one fluorescent dye, or (2) B-L1-D1-L2-D2, wherein B is a nucleobase, L1 and L2 are linkers such that at least one of L1 and L2 is an anionic linker, and D1 and D2 are members of a fluorescent donor/acceptor pair, such that one of D1 and D2 is a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto, and the other of D1 and D2 is an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response thereto.
Each anionic linker may contain one or more anionic groups, such as a sulfonic acid moiety, a sulfate monoester, an anionic phosphate, an anionic phosphonate, or a carboxylic acid. In one embodiment, L, L1 or L2 contains a phosphate diester moiety whose phosphorus atom is located within a chain of linker atoms (bridging position) or can be a substituent attached to a chain of linker atoms (non-bridging position). In another embodiment, the linker contains a monoanionic phosphonate ester which can be located within the linker chain or attached to the linker chain. Other embodiments are described further herein.
In embodiments in which the conjugate has the form B-L1-D1-L2-D2, one of L1 and L2 can be a nonanionic linker. In one embodiment, L1 is an anionic linker and L2 is non-anionic. For example, when L2 is non-anionic, D1-L2-D2 may comprise structure (a), (b) or (c) below:
(a) -D1-R21Z1C(O)R22R28-D2
(b) -D1-R28R22C(O)Z1R21-D2
(c) -D1-R28R22R28-D2
wherein: R21 is C1-C5 alkyldiyl, Z1 is NH, S, or 0, R22 is ethenediyl, ethynediyl, 1,3-butadienediyl, 1,3-butadiynediyl, a 5- or 6-membered ring having at least one unsaturated bond or a fused ring structure having at least one unsaturated bone, and R28 is a bond or spacer group (a linking segment) that links R22 to D1 or D2. In another embodiment, L1 can be a nonionic linker, of which the following are examples: xe2x80x94Cxe2x89xa1CCH2NHxe2x80x94, xe2x80x94Cxe2x89xa1CCH2NHC(O)(CH2)5NHxe2x80x94, xe2x80x94Cxe2x95x90CC(O)NH(CH2)5NHxe2x80x94, xe2x80x94Cxe2x89xa1CCH2OCH2CH2NHxe2x80x94, xe2x80x94Cxe2x89xa1CCH2OCH2CH2OCH2CH2NHxe2x80x94, xe2x80x94Cxe2x80x94Cxe2x80x94CH2OCH2CH2xe2x80x94NHxe2x80x94, and xe2x80x94Cxe2x89xa1C(p-C-4H6)OCH2CH2NHxe2x80x94.
Fluorescent dyes used in accordance with the invention can include any fluorescent compound suitable for the purposes of the present invention. Typically, each dye comprises a conjugated, resonance-delocalized or aromatic ring system that absorbs light at a first wavelength and emits light at a second wavelength in response thereto. For example, the dyes can be selected independently from any of a variety of classes of fluorescent compounds, such as xanthene, rhodamine, dibenzorhodamine, fluorescein, [8,9]benzophenoxazine, cyanine, phthalocyanine, squaraine, or bodipy dye.
In another aspect, the invention includes a labeled nucleoside triphosphate comprising a conjugate of the type described herein. In one embodiment, the labeled nucleoside triphosphate is not 3xe2x80x2-extendable. For example, the labeled nucleoside triphosphate can be a 2xe2x80x2,3xe2x80x2-dideoxynucleotide or 3xe2x80x2-fluoro-2xe2x80x2,3xe2x80x2-dideoxynucleotide. In another embodiment, the labeled nucleoside triphosphate is extendable and contains a 3xe2x80x2-hydroxyl group.
In another aspect, the invention includes a polynucleotide comprising a conjugate of the type discussed herein. In one embodiment, the conjugate is located in a 3xe2x80x2 terminal nucleotide subunit of a polynucleotide, such that the subunit may be extendable or nonextendable. In another embodiment, the conjugate is located on a non-terminal nucleotide subunit.
In a further embodiment, the invention provides a mixture comprising a plurality of different-sequence polynucleotides, wherein at least one polynucleotide contains a conjugate as described herein. In one embodiment, the mixture comprises at least two different-sequence polynucleotides which each contain a different conjugate to identify the attached polynucleotide.
In another embodiment, the mixture comprises four classes of polynucleotides, wherein the polynucleotides in each class terminate with a different terminator subunit type that contains a distinct nucleobase-dye conjugate to identify the polynucleotides in that class.
The invention also includes a method of identifying one or more polynucleotide(s). In the method, one or more labeled different-sequence polynucleotides are formed such that each different-sequence polynucleotide contains a unique conjugate as described herein. The one or more labeled different-sequence polynucleotides are separated by electrophoresis on the basis of size, and one or more different-sequence polynucleotides are identified on the basis of electrophoretic mobilities and fluorescence properties.
The invention also provides a method of forming a labeled polynucleotide strand, the method comprising reacting together (i) a duplex polynucleotide comprising a 3xe2x80x2-extendable strand hybridized to a complementary template strand having a 5xe2x80x2 overhang, (ii) a template-dependent polymerase enzyme, and (iii) a labeled nucleoside triphosphate containing a conjugate as described herein, under conditions effective to form a labeled polynucleotide containing the conjugate. In one embodiment, the labeled nucleoside triphosphate is nonextendable. In another embodiment, the labeled nucleoside triphosphate is extendable.
The invention also provides a method of sequencing a target polynucleotide sequence. In the method, four classes of polynucleotides are formed which are complementary to a target polynucleotide sequence, by template-dependent primer extension, wherein the polynucleotides in each class terminate with a different terminator subunit type that contains a distinct nucleobase-dye conjugate to identify the polynucleotides in that class. The resultant polynucleotides are separated on the basis of size to obtain a mobility pattern from which the sequence of the target polynucleotide sequence can be determined.
The invention also provides kits for performing the various methods of the invention. For nucleic acid sequencing, the kit comprises at least one labeled nucleoside triphosphate comprising a conjugate described herein. The kit may also include one or more of the following components: a 3xe2x80x2-extendable primer, a polymerase enzyme, one or more 3xe2x80x2 extendable nucleotides which are not labeled with conjugate, and/or a buffering agent. In some embodiments, the kit includes at least one labeled nucleoside triphosphate that is nonextendable. In other embodiments, the kit comprises four different labeled nucleoside triphosphates which are complementary to A, C, T and G, and each of which contains a distinct conjugate as described herein. In yet another embodiment, the labeled nucleoside triphosphates are nonextendable. In another embodiment, the labeled nucleoside triphosphates are extendable ribonucleoside triphosphates. In another embodiment, the kit comprises at least one labeled, nonextendable nucleoside triphosphate comprising a conjugate described herein, and one or more of the following components: a 3xe2x80x2-extendable primer, a polymerase enzyme, and/or a buffering agent.
These and other objects and features of the invention will become more apparent from the detailed description.